|Molecular Vision 2006;
Received 27 September 2005 | Accepted 4 January 2006 | Published 10 January 2006
A missense mutation in the γD-crystallin gene GRYGD associated with autosomal dominant congenital cataract in a Chinese family
Feng Gu,1,2 Rong
Li,3 Xi Xin Ma,3 Li Song Shi,2,4 Shang Zhi
Huang,2,4 Xu Ma1,2,5
1Department of Genetics, National Research Institute for Family Planning, Beijing, China; 2Peking Union Medical College, Beijing, China; 3Zhengzhou Er Qi District Hospital, Zhengzhou, Henan, China; 4Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China; 5Department of Reproductive Genetics, WHO Collaborative Center for Research in Human Reproduction, Beijing, China
Correspondence to: Xu Ma, National Research Institute for Family Planning, 12 Da-hui-si, Hai Dian, Beijing, 100081, China; Phone: +86-010-62176870; FAX: +86-010-62179059; email: email@example.com
Purpose: To identify the genetic defect in autosomal dominant congenital cataracts in a six generation Chinese family.
Methods: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. All the members were genotyped with microsatellite markers at loci which were considered to be associated with cataracts. A two-point LOD score was calculated using the Linkage package after genotyping. A mutation was detected by direct sequencing using gene specific primers.
Results: Clinical heterogeneity was observed within this family, three affected individuals showed nuclear cataract and others had coralliform cataracts. Significant evidence of linkage was obtained at markers D2S325 (LOD score [Z]=3.10, recombination fraction [θ]=0.0) and D2S1782 (Z=5.97, θ=0.0), respectively. Haplotype analysis indicated that the cataract gene was close to those two markers. Sequencing of the γD-crystallin gene (CRYGD) revealed a C>T transition in exon 2, that causes a conservative substitution of Arg to Cys at codon 14 (R14C). This mutation co-segregated with all affected individuals and was not observed in unaffected or 100 normal unrelated individuals. Bioinformatic analyses also showed that a highly conserved region was located at Arg14.
Conclusions: This study is the first reported case with phenotype of coralliform/nuclear cataract that associated with the mutation of Arg14Cys (R14C) CRYGD.
Hereditary cataract is a clinically and genetically heterogeneous lens disease that induced a significant proportion of visual impairment and blindness in childhood [1,2]. Over the past few years, mutations in several functionally diverse genes have now been identified in a small percentage of congenital cataract patients, which included: CRYAA , CRYAB , CRYBA1/A3 , CRYBB1 , CRYBB2 [7,8], CRYGC [9,10], CRYGD , GJA3 , GJA8 [13,14], MIP , BFSP2 [16,17], PITX3 , HSF4 , and LIM2 .
Even though the hereditary aspects of congenital cataracts have been recognized for nearly a century and some progress has been made, the relationship between morphology and genetic etiology of congenital cataract is not clear yet, this is due to the complex developmental process of the ocular lens and a wide variation in opacity morphology.
According to the specific morphology of the lens concerned with position and appearance, under slit lamp examination, congenital cataracts were classified as anterior polar, posterior polar, nuclear, lamellar (zonular), pulverulent, aculeiform, cerulean, cortical, polymorphic, sutural, coralliform, and total cataract .
In this study, we detected linkage of an autosomal dominant inherited coralliform/nuclear cataract with the D2S325 and D2S1782 markers in a six generation Chinese family. A missense mutation (C34T) in CRYGD was identified, which resulted in an Arg to Cys (R14C) substitution in CRYGD. To our knowledge, this is the first reported case with phenotype of coralliform/nuclear cataracts caused by C34T mutation in CRYGD.
Clinical evaluations and DNA specimens
The protocol for the study was approved by the Ethics Committee of our institute. Informed consent was obtained from all family members participating in this study. The family comprised 25 affected individuals from a six generation pedigree (Figure 1), originating from the province of Henan, China. The study consisted of 30 members, including 16 affected individuals, 7 unaffected individuals, and 7 spouses (Figure 1). Clinical and ophthalmological examinations were performed. The diagnosis of cataract was confirmed by ophthalmologists. There was no history of other ocular or systemic abnormalities in the family.
Exclusion analysis was performed by allele sharing of the microsatellite markers, which were linked with known cataract loci, on affected individuals. Genotyping was performed as described previously  using microsatellite markers D2S325 and D2S1782 at 2q33-35, which are linked with the coralliform cataract. The oligonucleotide primer sequences were taken from NCBI and GDB.
A two-point LOD score (Z) was calculated using the MLINK subprogram of the Linkage package (version 5.1). The mode of inheritance was considered to be autosomal dominant with full penetrance. The gene frequency was set at 1/10,000. As the allelic frequencies of the polymorphic markers are unknown in the Chinese population, they were considered to be equally distributed.
Mutations in CRYGA, CRYGB, CRYGC, and CRYGD were screened by direct sequencing. PCR products of the three exons and flanking intron sequences of CRYGA, CRYGB, CRYGC, and CRYGD  were sequenced on an ABI A377 Automated Sequencer (PE Biosystems, Foster City, CA).
Denaturing (D)HPLC was used to screen the mutation identified in the patients, on remaining patients, family members, and 100 normal control subjects in exon 2 of the CRYGD gene using a commercial system (Wave DHPLC; Transgenomic, San Jose, CA). Gene specific PCR primers were used to amplify the fragment which harbored the point mutation. DHPLC was performed as follows: initial concentration at 48% of buffer A (0.1 M triethylammonium acetate-TEAA; Transgenomic), and 52% of buffer B (0.1 M TEAA containing 25% acetonitrile; Transgenomic) at 65 °C.
The proband was a 17-year-old male (IV:9) who had a bilateral cataract in 1993. The form of the opacification was irregular as sea coral, with crystal clumps radiating from center to capsule. Except for affected individuals IV:1, IV:2, and V:16 with nuclear opacification, all the affected individuals showed a phenotype of coralliform cataract (age of VI:1 was 5 and IV:7 was 38; Figure 2A,B). Slit-lamp photographs of the affected individuals (IV:1, IV:5, V:16; the age of IV:1 was 54; Figure 2C) showed that opacities were located mainly in the nuclear areas of both lenses and there were pulverulent opacities in the perinuclear areas, which are different from the coralliform cataract. Several of the affected individuals had high myopia with elongated axis oculi; ocular lengths were (OD/OS) 29.98 mm/30.54 mm, 27.58 mm/28.61 mm, 29.02 mm/29.66 mm, 24.1 mm/24.5 mm in individuals IV:1, IV:5, V:4, and VI:1, respectively. No systemic or other ocular anomalies were observed in the patients.
Linkage and haplotype analysis
Allele-sharing analysis excluded the linkage of the disease in the family with all known loci of cataract except that for CRYGs, D2S325, and D2S1782 at 2q33 (data not shown). Haplotype analysis showed that the affected individuals in the family shared a common haplotype with markers D2S325 and D2S1782 at 2q33 (Figure 1). Significant evidence of linkage was observed with microsatellite markers D2S325 (LOD score [Z]=3.10, at recombination fraction [θ]=0.0) and D2S1782 (Z=5.97, at θ=0.0), respectively (Table 1). This implied that one of the members of CRYG in the region might be responsible for the disease.
Mutation detection for CRYGA, CRYGB, CRYGC, and CRYGD
Direct cycle sequencing of the amplified fragments of CRYGs in two affected individuals identified a single base alteration C34T (Figure 3A) in exon 2 of CRYGD (NM_006891), which resulted in a substitution of Arg to Cys at codon 14 (R14C). Denaturing HPLC analysis confirmed this mutation, which co-segregated with all affected individuals in the family, and were not observed in any of the unaffected family members or 100 normal controls. The remainder of the coding sequence did not show any sequence change.
Multiple-sequence alignment and mutation analysis
Using the NCBI and UCSC websites, we obtained multiple-sequence alignment of the CRYG family proteins in various species with DNAMAN biosoftware, including Homo sapiens, Canis familiaris, Mus musculus, and Rattus norvegicus (Figure 3B). We found that codon 14, where mutation (R14C) occurred, located within a highly conserved region.
Furthermore, we used online bioinformatics software SIFT  to predict whether the amino acid substitution in CRYGD could have a phenotypic effect, with a result of the substitution at position 14 from R to C is predicted to affect protein function with a score of 0.00. While positions with normalized probabilities less than 0.05 are predicted to be deleterious, those greater than or equal to 0.05 are predicted to be tolerated. All of these indicated that the Arg-14 is an important residue for the function of CRYGD.
Coralliform cataract is an uncommon form of congenital cataract, which was named for the morphological resemblance of these lens opacities to sea coral, extending from the nucleus into the anterior and posterior cortex. It was first reported in 1895  and subsequently described as an autosomal dominant trait in three British pedigrees circa in 1910 . Since then, this phenotype has been seldom reported. Phenotypes of aceuliform and fasciculiform cataract are also associated with needle-like projections extending from the nucleus into the anterior and posterior cortex, there may well be overlap among the three phenotypes. Some results [9,23,27,28] showed that the mutations of CRYGD, locating on 2q33-35, were responsible for coralliform, aceuliform, and fasciculiform phenotype, respectively. Recently, a new locus for coralliform cataract has been mapped to chromosome 2p24-pter in a four generation Chinese family . There are, therefore, two genetic loci reported to date for coralliform cataract at 2q33-35 and 2p24-pter.
In this study, according to the phenotype of congenital cataracts, members of the family were firstly genotyped with microsatellite markers at all known loci of congenital cataracts, and special attention was paid to markers on 2q33-35 as there was allele sharing. Haplotype analysis showed that the affected individuals in this family shared a common haplotype with two markers, D2S325 and D2S1782, within the region of 2q33-35, where the members of the CRYG family locate.
The γ-crystallin gene cluster comprises six genes; CRYGA, CRYGB, CRYGC, CRYGD, CRYGE, and CRYGF. In mammals, each of these genes consists of three exons, only CRYGC and CRYGD encode abundant lens γ-crystallins in human [30,31]. CRYGE and CRYGF are pseudogenes with in-frame stop codons. CRYGC/D is one of the only two γ-crystallins to be expressed at high concentrations in the fiber cells of the embryonic human lens, which subsequently forms lens nucleus fibers. For this reason and with the phenotype, we focus our attention on CRYGD. After screening for mutations in CRYGA, CRYGB, CRYGC, and CRYGD by direct cycle sequencing, we identified a C>T transition in exon 2 of GRYGD, which presented only in affected members of the family. The transition C34T locates in exon 2, which was predicted to cause a conservative substitution of Arg to Cys at codon 14 (R14C). The results of multiple-sequence alignment and mutation analysis also confirmed the importance of Arg14 for the function of CRYGD.
Mutations in the γD-crystallin encoding gene (CRYGD) have been demonstrated to be one of the most frequent reasons for isolated congenital cataracts. Mutations within CRYGD so far reported were responsible for several phenotypes; progressive juvenile-onset punctate (R14C) , cerulean (P23T) , prismatic (R36S) , aceuliform (R58H) [9,27], nuclear (W156X) , lamellar (P23T) , fasciculiform (P23T) , and coralliform cataract (P23T) [23,27]. Functional studies on the mutation of CRYGD polypeptides in vitro also have shown that polypeptides with R14C, P23T, and R58H mutations are less soluble and more prone to crystallization [35-37]. All these results revealed that the CRYGD plays an important role in maintaining lens transparency.
Stephan et al.  have reported a C>T mutation at nucleotide 34 of CRYGD exon 2 in a family with juvenile-onset punctate cataract, which was identical to our study. Compared with affected individuals in that family (progressive trait), there was not enough evidence to prove that the progressive trait of the affected individuals in this Chinese family was not due to long-term follow-up. Meanwhile, there was no obvious progressive development in this Chinese family since the two affected individuals at different ages have similar lens opacities (Figure 2). The difference of phenotype cataract between the two families was significant; punctate cataract in Stephan's case, but coralliform/nuclear form in this report. The discrepancy is consistent with the fact that the relationship between morphology and genetic etiology of congenital cataract was not reliable in different populations [7,9,11,23,24,27,28,32-34,38].
In this family, there are two different phenotypes, coralliform and nuclear cataracts. Intrafamilial variation implied that clinically differentiation of both types of opacifications could be attributed to the action of other modifier genes.
High myopia is frequently seen in affected individuals, who have elongated ocular axis, suggesting that the mutation in this family may play a role in the development of high myopia. Further studies need to provide further insights into the molecular pathology of high myopia in this family.
Allele sharing analysis was a quick procedure for excluding a reported locus responsible for the disease in a family under study. If no allele sharing has been detected in affected individuals, there could be no linkage with the locus. Otherwise, a further linkage analysis was required for confirmation since shared alleles could come from the unaffected parent. Under linkage analysis a higher LOD score has been obtained at marker D2S1782 (Z=5.97) but not at D2S325 (3.10) in this family, while D2S325 lies more closely to the disease gene (Figure 1). The reason for it was that more individuals were homozygous for D2S325 in the family than that for D2S1782.
This study is the first example of Intrafamilial variability in the cataract phenotype associated with R14C CRYGD mutation. The clinical variability within the family or between the two families can be attributed to the action of other modifier genes or, perhaps less likely, environmental factors. In summary, our results provided evidence for clinical heterogeneity of punctate, coralliform, and nuclear cataracts. Our results further confirmed that CRYGD is important in the maintenance of optical clarity.
The authors thank the family for their participation in this project and Dr. Siquan Zhu (Beijing Tongren Hospital, Capital University of Medical Sciences, Beijing), Dr. Guangying Zheng (the First affiliated Hospital, Zhengzhou University) for phenotype identification, Dr. Xuemin Jin for sample collection, and Dr. Xiaohui Yang for photographic records. This work is partly supported by the National "973" Basic Research Funding Scheme of China (grant number 2001CB5103) and National Infrastructure Program of Chinese Genetic Resources (2004DKA30490).
1. Scott MH, Hejtmancik JF, Wozencraft LA, Reuter LM, Parks MM, Kaiser-Kupfer MI. Autosomal dominant congenital cataract. Interocular phenotypic variability. Ophthalmology 1994; 101:866-71.
2. Hejtmancik JF. The genetics of cataract: our vision becomes clearer. Am J Hum Genet 1998; 62:520-5.
3. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998; 7:471-4.
4. Berry V, Francis P, Reddy MA, Collyer D, Vithana E, MacKay I, Dawson G, Carey AH, Moore A, Bhattacharya SS, Quinlan RA. Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Am J Hum Genet 2001; 69:1141-5.
5. Kannabiran C, Rogan PK, Olmos L, Basti S, Rao GN, Kaiser-Kupfer M, Hejtmancik JF. Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene. Mol Vis 1998; 4:21 <http://www.molvis.org/molvis/v4/a21/>.
6. Mackay DS, Boskovska OB, Knopf HL, Lampi KJ, Shiels A. A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am J Hum Genet 2002; 71:1216-21.
7. Litt M, Carrero-Valenzuela R, LaMorticella DM, Schultz DW, Mitchell TN, Kramer P, Maumenee IH. Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum Mol Genet 1997; 6:665-8.
8. Vanita, Sarhadi V, Reis A, Jung M, Singh D, Sperling K, Singh JR, Burger J. A unique form of autosomal dominant cataract explained by gene conversion between beta-crystallin B2 and its pseudogene. J Med Genet 2001; 38:392-6.
9. Heon E, Priston M, Schorderet DF, Billingsley GD, Girard PO, Lubsen N, Munier FL. The gamma-crystallins and human cataracts: a puzzle made clearer. Am J Hum Genet 1999; 65:1261-7.
10. Ren Z, Li A, Shastry BS, Padma T, Ayyagari R, Scott MH, Parks MM, Kaiser-Kupfer MI, Hejtmancik JF. A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract. Hum Genet 2000; 106:531-7.
11. Stephan DA, Gillanders E, Vanderveen D, Freas-Lutz D, Wistow G, Baxevanis AD, Robbins CM, VanAuken A, Quesenberry MI, Bailey-Wilson J, Juo SH, Trent JM, Smith L, Brownstein MJ. Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc Natl Acad Sci U S A 1999; 96:1008-12.
12. Mackay D, Ionides A, Kibar Z, Rouleau G, Berry V, Moore A, Shiels A, Bhattacharya S. Connexin46 mutations in autosomal dominant congenital cataract. Am J Hum Genet 1999; 64:1357-64.
13. Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998; 62:526-32.
14. Polyakov AV, Shagina IA, Khlebnikova OV, Evgrafov OV. Mutation in the connexin 50 gene (GJA8) in a Russian family with zonular pulverulent cataract. Clin Genet 2001; 60:476-8.
15. Berry V, Francis P, Kaushal S, Moore A, Bhattacharya S. Missense mutations in MIP underlie autosomal dominant 'polymorphic' and lamellar cataracts linked to 12q. Nat Genet 2000; 25:15-7.
16. Conley YP, Erturk D, Keverline A, Mah TS, Keravala A, Barnes LR, Bruchis A, Hess JF, FitzGerald PG, Weeks DE, Ferrell RE, Gorin MB. A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2. Am J Hum Genet 2000; 66:1426-31.
17. Jakobs PM, Hess JF, FitzGerald PG, Kramer P, Weleber RG, Litt M. Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. Am J Hum Genet 2000; 66:1432-6.
18. Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS, Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet 1998; 19:167-70.
19. Bu L, Jin Y, Shi Y, Chu R, Ban A, Eiberg H, Andres L, Jiang H, Zheng G, Qian M, Cui B, Xia Y, Liu J, Hu L, Zhao G, Hayden MR, Kong X. Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataract. Nat Genet 2002; 31:276-8.
20. Pras E, Frydman M, Levy-Nissenbaum E, Bakhan T, Raz J, Assia EI, Goldman B, Pras E. A nonsense mutation (W9X) in CRYAA causes autosomal recessive cataract in an inbred Jewish Persian family. Invest Ophthalmol Vis Sci 2000; 41:3511-5.
21. Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic basis of inherited cataract and associated phenotypes. Surv Ophthalmol 2004; 49:300-15.
22. Qi Y, Jia H, Huang S, Lin H, Gu J, Su H, Zhang T, Gao Y, Qu L, Li D, Li Y. A deletion mutation in the betaA1/A3 crystallin gene (CRYBA1/A3) is associated with autosomal dominant congenital nuclear cataract in a Chinese family. Hum Genet 2004; 114:192-7.
23. Mackay DS, Andley UP, Shiels A. A missense mutation in the gammaD crystallin gene (CRYGD) associated with autosomal dominant "coral-like" cataract linked to chromosome 2q. Mol Vis 2004; 10:155-62 <http://www.molvis.org/molvis/v10/a21/>.
24. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31:3812-4.
25. Gunn RM. Peculiar coralliform cataract with crystals in the lens. Trans Ophthalmol Soc 1895; XV:119.
26. Harman NB. Ten pedigrees of congenital and infantile cataract; lamellar, coralliform, discoid, and posterior polar with microphthalmia. Trans Ophthalmol Soc U K 1910; 30:251-74.
27. Zenteno JC, Morales ME, Moran-Barroso V, Sanchez-Navarro A. CRYGD gene analysis in a family with autosomal dominant congenital cataract: evidence for molecular homogeneity and intrafamilial clinical heterogeneity in aculeiform cataract. Mol Vis 2005; 11:438-42 <http://www.molvis.org/molvis/v11/a51/>.
28. Shentu X, Yao K, Xu W, Zheng S, Hu S, Gong X. Special fasciculiform cataract caused by a mutation in the gammaD-crystallin gene. Mol Vis 2004; 10:233-9 <http://www.molvis.org/molvis/v10/a29/>.
29. Gao L, Qin W, Cui H, Feng G, Liu P, Gao W, Ma L, Li P, He L, Fu S. A novel locus of coralliform cataract mapped to chromosome 2p24-pter. J Hum Genet 2005; 50:305-10.
30. Russell P, Meakin SO, Hohman TC, Tsui LC, Breitman ML. Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens. Mol Cell Biol 1987; 7:3320-3.
31. Brakenhoff RH, Aarts HJ, Reek FH, Lubsen NH, Schoenmakers JG. Human gamma-crystallin genes. A gene family on its way to extinction. J Mol Biol 1990; 216:519-32.
32. Nandrot E, Slingsby C, Basak A, Cherif-Chefchaouni M, Benazzouz B, Hajaji Y, Boutayeb S, Gribouval O, Arbogast L, Berraho A, Abitbol M, Hilal L. Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts. J Med Genet 2003; 40:262-7.
33. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novak P, Rezacova P, Ondrova L, Filipec M, Sedlacek J, Elleder M. Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Hum Mol Genet 2000; 9:1779-86.
34. Santhiya ST, Shyam Manohar M, Rawlley D, Vijayalakshmi P, Namperumalsamy P, Gopinath PM, Loster J, Graw J. Novel mutations in the gamma-crystallin genes cause autosomal dominant congenital cataracts. J Med Genet 2002; 39:352-8.
35. Pande A, Pande J, Asherie N, Lomakin A, Ogun O, King JA, Lubsen NH, Walton D, Benedek GB. Molecular basis of a progressive juvenile-onset hereditary cataract. Proc Natl Acad Sci U S A 2000; 97:1993-8.
36. Pande A, Annunziata O, Asherie N, Ogun O, Benedek GB, Pande J. Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin. Biochemistry 2005; 44:2491-500.
37. Evans P, Wyatt K, Wistow GJ, Bateman OA, Wallace BA, Slingsby C. The P23T cataract mutation causes loss of solubility of folded gammaD-crystallin. J Mol Biol 2004; 343:435-44.
38. Gill D, Klose R, Munier FL, McFadden M, Priston M, Billingsley G, Ducrey N, Schorderet DF, Heon E. Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2. Invest Ophthalmol Vis Sci 2000; 41:159-65.